Bibliographic details of the publications referred to by author in this specification are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined following the bibliography. A summary of the SEQ ID NOs. is provided before the Examples.
Apoptosis, the physiologic and genetically modulated process of cell death, is of central importance for modelling tissues and maintaining homeostasis in multicellular organisms (Kerr et al., 1972; Jacobson et al., 1997). Great progress is being made towards understanding the biochemistry underlying this intrinsic suicide program. The cellular apoptotic effector molecules include a set of cysteine proteinases, termed caspases, that degrade critical cellular substrates (Nicholson and Thornberry, 1997). The regulatory machinery that governs the activation of the caspases is less well understood. However a family of proteins of which Bcl-2 is the prototypic molecule (and is referred to as the Bcl-2 family of proteins) plays a central role (Jacobson, 1997; Reed, 1997; Kroemer, 1997).
Bcl-2 was the first intracellular regulator of apoptosis to be identified (Vaux et al., 1988) and high levels enhance cell survival under diverse cytotoxic conditions. Other cellular homologues, such as Bcl-xL (Boise et al., 1993) and Bcl-w (Gibson et al., 1996), also enhance cell survival, as do more distantly related viral homologues, such as the adenovirus E1B 19K protein (White et al., 1992) and Epstein-Barr virus BHRF-1 (Henderson et al., 1993). However, the family also includes members such as Bax (Oltvai et al., 1993) and Bak (Chittenden et al., 1995; Kiefer et al., 1995; Farrow et al., 1995), which antagonise the activity of the pro-survival proteins and provoke apoptosis when expressed at high concentrations. The relative concentrations of the opposing subfamily members may determine whether the cell lives or dies (Oltvai et al., 1993).
The homology between members of the Bcl-2 family is greatest within four small regions, designated Bcl-2 Homology (BH) regions (Yin et al., 1994; Borner et al., 1994; Chittenden et al., 1995; Gibson et al., 1996; Zha et al., 1996). The N-terminal BH4 domain is restricted to some antagonists of apoptosis, while BH1, BH2 and BH3 can be found in both sub-families (reviewed by Kroemer, 1997). In the tertiary structure determined for Bcl-xL, the BH1, BH2 and BH3 domains form an elongated hydrophobic cleft on the surface of the molecule. stabilised by the BH4 amphipathic helix (Muchmore et al., 1996; Sattler et al., 1997). Most members of the Bcl-2 family contain a C-terminal hydrophobic region, which appears to be important for their localisation to intracytoplasmic membranes (reviewed by Kroemer, 1997).
Protein interactions are an important feature of the Bcl-2 family. Interaction between the pro-survival and pro-apoptotic proteins, such as Bcl-2 with Bax or Bak, requires the BH1 and BH2 domains of the former (Yin et al., 1994; Sedlak et al., 1995; Hanada et al., 1995) and the BH3 domain of the latter (Chittenden et al., 1995; Zha et al., 1996). BH3 peptides bind to the hydrophobic cleft of Bcl-xL formed by BH1, BH2 and BH3 (Sattler et al., 1997). Although mutagenesis of Bcl-2 and Bc1-xL initially suggested that their ability to inhibit cell death required binding to a pro-apoptotic family member (Yin et al., 1994), Bc1-xL mutants have, been identified that do not bind Bax or Bak but still block apoptosis (Cheng et al., 1996).
An additional group of pro-apoptotic proteins has recently been described. Bik/Nbk (Boyd et al., 1995; Zha et al., 1996), Bid (Wang et al., 1996) and Hrk (Inohara et al., 1997). The only feature they share in common with each other, or the Bcl-2 family, is the small (9 amino acid) BH3 domain.
This region is essential for the ability of these proteins to promote cell death.
In work leading up to the present invention, the inventors have identified a novel member of the Bcl-2 family, designated herein “Bim”. In accordance with the present invention. Bim induces cell death and acts as a “death-ligand” for certain members of the pro-survival Bcl-2 family. The identification of this new gene permits the identification and rational design of a range of products for use in therapy, diagnosis, antibody generation and involving modulation of physiological cell death. These therapeutic molecules may act as either antagonists or agonists of Bim's function and will be useful in cancer autoimmune or degenerative disease therapy.